Specific DNA recognition by the type II restriction endonuclease MunI:: The effect of pH
Specific DNA recognition by the type II restriction endonuclease MunI:: The effect of pH
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DOI：
10.1021/bi0113566
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发表时间：
2001-12-11
期刊：
影响因子：
2.9
通讯作者：
Ladbury, JE
中科院分区：
文献类型：
--
作者：
Haq, I;O'Brien, R;Ladbury, JE
为了研究pH对序列特异性结合的影响，进行了蛋白质MunI与特异性和非特异性寡核苷酸的相互作用的热力学表征。MunI是一种II型限制性内切核酸酶，其能够特异性结合，但在不存在镁离子的情况下失去其酶活性。在10和25 degreesC的特异性和非特异性相互作用的比较表明，后者伴随着焓的小的变化，和恒定压力热容的变化可以忽略不计。在25 ℃下通过pH 5.75-9.0范围时，特异性复合物形成的亲和力降低20倍。这种相互作用伴随着假定位于蛋白质上的基团的质子化。基于拟合数据的最简单模型，观察到两个不同的质子化事件。在低pH值下，每个蛋白质分子中有两个基团发生质子化，pK（a）分别为6.0和6.9。在高pH值，进一步独立的质子化发生涉及两个基团的pK（a）值为8.9和约10.7的自由和结合形式，分别。从pH 6.5到8.5，热容的变化范围为-2.7到-1.7 kJ mol（-1）K-1。热容变化的这一范围可以由相互作用分子的质子化效应来解释。热容量的变化，计算表面积埋藏使用以前建立的关系（1.15 kJ mol（-1）K-1），不相关的实验确定的值。
To investigate the effect of pH on sequence-specific binding, a thermodynamic characterization of the interaction of the protein MunI with a specific, and a nonspecific, oligonucleotide was performed. MunI is a type II restriction endonuclease which is able to bind specifically, but loses its enzymatic activity in the absence of magnesium ions. Comparison of the specific and nonspecific interactions at 10 and 25 degreesC shows that the latter is accompanied by a small change in enthalpy, and a negligible change in constant pressure heat capacity. On going through the pH range 5.75-9.0 at 25 degreesC, the affinity of specific complex formation is reduced by 20-fold. The interaction is accompanied by the protonation of groups assumed to be on the protein. Based on the simplest model that will fit the data, two distinct protonation events are observed. At low pH, two groups per protein molecule undergo protonation with a pK(a) of 6.0 and 6.9 in the free and bound forms, respectively. At high pH, a further independent protonation occurs involving two groups with pK(a) values of 8.9 and approximately 10.7 in the free and bound forms, respectively. The change in heat capacity ranges from -2.7 to -1.7 kJ mol(-1) K-1 in going from pH 6.5 to 8.5. This range of variation of change in heat capacity can be accounted for by the effects of protonation of the interacting molecules. The change in heat capacity, calculated from surface area burial using a previously established relationship (1.15 kJ mol(-1) K-1), does not correlate well with the experimentally determined values.